- Volume 139, Issue 3, 1993

The association of staphylococci with the mucus gel that overlays the mucosa of the respiratory tract may lead to clearance of cocci or, in certain conditions such as cystic fibrosis (CF), to colonization. In the present study, a quantitative radioassay was used to study the effect of Ca2+, which is elevated in CF sputa, on the adhesion of 3H-labelled Staphylococcus aureus to submaxillary gland mucin immobilized in MaxiSorp 96-well, break-apart modules. Ca2+ significantly enhanced the adhesion of S. aureus (five strains) and Staphylococcus epidermidis (four strains). The reaction was specific because adhesion was not enhanced in the presence of Mg2+, Ca2+ + EGTA (a Ca2+ chelator) or protamine and was not attributable to hydrophobicity of the test strains. Staphylococcal adhesion was significantly (P⩽0·005) blocked in the presence of highly sialated and sulphated reagents, which suggests that Ca2+ binds to the sialic acid and sulphate residues of immobilized mucin. The Ca2+ -binding sites on the surface of S. aureus were trypsin-sensitive; in addition, 125I-labelled solubilized S. aureus surface proteins reacted with immobilized mucin in a direct binding assay, and the reaction was significantly enhanced by Ca2+. Autoradiography demonstrated that 45Ca bound directly to two polypeptides (M r 170000 and 150000) of solubilized staphylococcal surface proteins separated by SDS-PAGE, and that 125I-labelled mucin bound directly to three staphylococcal polypeptides (M r 40000, 35000, and 29000). These results suggest that S. aureus adhesion to mucin is mediated by at least two mechanisms: via Ca2+-binding surface proteins in the presence of Ca2+ and via mucin-binding surface proteins unrelated to Ca2+.

The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3–4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.

Eikenella corrodens is emerging as an important human pathogen, in both extra-oral and periodontal infections. From a clone bank of Eikenella corrodens chromosomal DNA produced in Escherichia coli JM109, twenty-two clones expressed Eikenella antigens and of these, two expressed functional haemagglutinins. By virtue of different restriction maps and a lack of homology by Southern hybridization, the two cloned fragments encoding the two haemagglutinins have been shown to be distinct. Maxicell analysis revealed that clone 1, carrying plasmid pVKR201, produces three Eikenella proteins, one of 31·5 kDa and two of approximately 14 kDa each. Expression of each of the proteins appears to be under the control of an Eikenella promoter(s). Clone 2, carrying plasmid pVKR301, produces two proteins, one of 93 kDa and the second of 17 kDa. Expression of both of these proteins in E. coli requires the lac promoter in the vector. By preparing a series of subclones and testing each by maxicell analysis and for haemagglutination activity, a functional map of the insert of clone 1 was deduced and the 31·5 kDa polypeptide identified as the haemagglutinin. Using similar methods, the 17 kDa protein was found to be the haemagglutinin of clone 2. The nucleotide sequences of both haemagglutinin genes were determined and are presented. Computer analysis revealed no homology between the two haemagglutinins, and no homology to any previously sequenced proteins. These are the first genes of this genus to be cloned and sequenced.

Eikenella corrodens is a Gram-negative microaerophilic rod which is gaining recognition as an important human pathogen. We have previously reported the cloning and expression in Escherichia coli of a 3·6 kb Eik. corrodens genomic DNA fragment which encodes a 31·5 kDa haemagglutinin. Maxicell analysis revealed that this fragment also encodes two proteins of approximately 14 kDa. Nucleotide sequencing of the 2·2 kb fragment upstream of the haemagglutinin gene revealed two open reading frames with strong homology to genes encoding pilin subunit proteins of the type 4 or N-methylphenylalanine class. The two pilin genes, ecpA and ecpB, are complete and are expressed in E. coli. Southern analysis of ten additional Eik. corrodens strains revealed that all possess fragments homologous to ecpA. These data represent the first molecular evidence for pili in E. corrodens.

Alginate production by the highly alginate-producing Pseudomonas aeruginosa 8821M was maximal at a dissolved oxygen tension (DOT) of 5% of air saturation. Lower DOT limited growth and alginate synthesis. At higher DOT values up to 70% of air saturation, the specific alginate production rate decreased. Nevertheless, the molecular mass of the alginate increased at higher aerations, as indicated by the viscosity of solutions of the isolated biopolymer. The specific activity of the four enzymes leading to GDP-mannuronic acid formation, phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP) and GDP-mannose dehydrogenase (GMD), increased with DOT of up to 25%. At higher DOT, however, only GMP and GMD maintained their maximum values. Changes observed at high oxygen concentrations in the relative activities of PMI and GMP, which are activities of the same bifunctional protein, were attributed to the much higher sensitivity of PMI activity to irreversible oxidative inactivation. The less pronounced decrease of PMM activity at high DOT correlated with an intermediate sensitivity to oxidative inactivation, but could also be related to sequential induction of PMM by the product of the PMI reaction. Thus, oxygen-dependence of alginate synthesis was at least partially the effect of DOT on GDP-mannuronic acid formation. Optimal aerations for maximal alginate production (DOT = 5–10%) were below the aeration level (70%) that led to the highest viscosity. These results suggest that, like GMD, polymerization activity is not very sensitive to oxidative inactivation and they are consistent with the hypothesis that polymerization is dependent on GMD activity, or is regulated in a similar way.

We have devised a convenient procedure to induce the yeast-to-mycelium transition of Yarrowia lipolytica in conditions which avoid the occurrence of the reverse process during the period of study. Yeast cells in late exponential phase were resuspended in water and cooled down to 4 °C for at least 15 min, then heat-shocked by inoculation into a pre-warmed (30 °C) medium containing N-acetyl-d-glucosamine. Under these conditions, yeast cells developed into large branching filaments which continued elongating for more than 24 h. Further, ornithine decarboxylase (ODC) activity and polyamine cell pools increased compared to those of cells maintained in glucose medium, which continued yeast-like growth. Addition of ODC inhibitors blocked mycelial development, but only if added during a critical initial period after which they had no effect. At effective concentrations, ODC inhibitors had no significant effect on cell growth. Comparative studies of intact and permeabilized cells suggest that this selective effect is probably due to the location of ODC in more than one cell compartment, one of them being inaccessible to the drugs. Blocking of the morphological transition by ODC inhibitors was specifically reversed by putrescine, and by growing the cells in the presence of 5-azacytidine. It is suggested that the effect of the latter compound is related to its capacity to inhibit DNA methylation, indicating a relationship between polyamines and DNA methylation at the onset of the differentiation process.

Exponential phase cells of Saccharomyces cerevisiae treated with the superoxide free-radical generating agent menadione (MD; 0·2 mm) for 60 min adapted to become resistant to the lethal effects of a higher concentration of MD (4 mm). Inhibition of protein synthesis by treatment with cycloheximide totally prevented the adaptation to MD, indicating that this is an inducible response completely dependent on protein synthesis; this differs from the situation with peroxide in which only some of the adaptive response is cycloheximide-sensitive. Cells subjected to heat shock (23 to 37 °C) or treatment with hydrogen peroxide (H2O2; 0·2 mm, 60 min) became more resistant to 4 mm-MD; however, MD pretreatment did not induce any thermotolerance or resistance to peroxide. These differences between the response to MD and H2O2 were reflected in the results of l-[35S]methionine labelling studies. Using one-dimensional electrophoresis, only one polypeptide (60 kDa) was seen to be induced by 0·2 mM–MD and this was also induced by heat shock but not peroxide stress. With heat shock or peroxide treatment the induction of at least 10 polypeptides was detected using this approach. Using an isogenic petite strain, it was found that functional mitochondria were needed for conferring full resistance to MD, but that induction of the adaptive response was not dependent on mitochondrial function.

A method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.